Automated whole breast ultrasound scanners of the latest generation have reached a level of comfortable application and high quality volume acquisition. Nevertheless, there is a lack of data concerning this technology. We investigated the diagnostic performance and inter-observer concordance of the Automated Breast Volume Scanner (ABVS) ACUSON S2000™ and questioned its implications in breast cancer diagnostics.We collected 100 volume data sets and created a database containing 52 scans with no detectable lesions in conventional ultrasound (BI-RADS®-US 1), 30 scans with benign lesions (BI-RADS®-US 2) and 18 scans with breast cancer (BI-RADS®-US 5).Two independent examiners evaluated the ABVS data on a separate workstation without any prior knowledge of the patients’ histories.The inter-rater reliability reached fair agreement : 0.19-0.53). With respect to the true category, the conditional inter-rater validity coefficient was κ=0.18 (95% CI: 0.00-0.26) for the benign cases and κ=0.80 (95% CI: 0.61-1.00) for the malignant cases.Combining the assessments of examiner 1 and examiner 2, the diagnostic accuracy (AC), sensitivity (SE) and specificity (SP) for the experimental ABVS were AC = 79.0% (95% CI: 67.3-86.1), SE = 83.3% (95% CI: 57.7-95.6) and SP = 78.1% (% CI: 67.3-86.1), respectively.However, after the ABVS examination, there were a high number of requests for second-look ultrasounds in up to 48.8% of the healthy women due to assumed suspicious findings in the volume data.In an exploratory analysis, we estimated that an ABVS examination in addition to mammography alone could detect a relevant number of previously occult breast cancers .The ABVS is a reliable imaging method for the evaluation of the breast with high sensitivity and a fair inter-observer concordance. However, we have to overcome the problem of the high number of false-positive results. Therefore, further prospective studies in larger collectives are necessary to define standard procedures in image acquisition and interpretation. Nevertheless, we consider the ABVS as being suitable for integration into breast diagnostics as a beneficial and reliable imaging method. Today, we can expect that more than one million women will be newly diagnosed with breast cancer each year . The mosBreast cancer screening programs are based on radiologic examinations of the breast. Additional imaging modalities are only indicated if suspicious or unclear findings are present in the mammogram. Mammography has demonstrated excellent sensitivity, specificity and inter-observer concordance ,6. NeverSo far, breast ultrasound is the most commonly accepted and reliable diagnostic method for women with clinically or radiologically suspicious breast lesions . FurtherIn the current report, we present data concerning an up-to-date technology in automated ultrasound, the Automated Breast Volume Scanner . The ABVS reconstructs 3D data sets of the entire breast volume from automatically acquired B-mode images. These data can be stored and analyzed on a separate workstation.We evaluated whether or not breast lesions, previously detected by means of conventional ultrasound, could also be detected and correctly classified by independent examiners who only used ABVS data. Furthermore, we analyzed the inter-observer concordance and performed a model calculation to scrutinize the potential implications of the ABVS in a screening setting.Our study was carried out at the Franziskus Hospital Breast Cancer Center in Bielefeld, Germany, between March 2010 and July 2011. For the ultrasound examinations, we used the Siemens ACUSON S2000™ ultrasound system with the integrated ABVS . Patients are generally referred to our outpatient department on account of specific diagnostic queries, such as palpable breast lesions, breast pain, suspicious mammograms and intensified screening in low-risk and high-risk populations. Usually, patients receive a clinical examination, mammography and conventional breast ultrasound as the standard diagnostic methods, as well as subsequent examinations whenever necessary. Additionally, we offer an optional ABVS examination to all our patients within the routine practice of our breast cancer center. As a diagnostic standard, ultrasound pictures are categorized according to the Breast Imaging Reporting and Data System criteria of the American College of Radiology (ACR BI-RADS®-US) . Study pAll data were obtained using a standard of care clinical protocol and approved equipment. Therefore, the responsible ethics committee did not demand additional approval for this non-interventional case study. Although the requirements of individual informed consent were waived, we decided that all study participants signed an additional informed consent form before the ABVS examination.Two independent examiners evaluated the cases from the anonymized ABVS database. We compared the performance of these two examiners  with each other and with the results from the conventional ultrasound (gold standard).For our study we used the ACUSON S2000™ ABVS , an ultrasound system that automatically acquires full-field volumes of the breast Figure . The sysConventional B-mode ultrasound examinations were performed by the author SW, a DEGUM  level II certified senior consultant in gynecology with 8 years’ experience in breast ultrasound . This exAfter having completed these diagnostic steps, eligible patients were additionally examined using the ABVS.According to the national regulatory authority statutes, breast US systems have to fulfill basic technical requirements and undergo regular quality control measures . These sThe author SW performed the experimental ABVS examination in order to create 3D data sets. For the ABVS examination, the patients were in the supine position with the ipsilateral hand placed on the head. Depending on the size of the breast, the examiner chose the number of scans to be taken from each side. Usually, breasts with a bra cup size A or B can be fully displayed by performing two volume scans  and AF (examiner 2) performed the independent interpretation of the ABVS data sets. Both, SG and AF, are DEGUM level I certified senior residents in gynecology with 5 years’ experience in breast ultrasound .The independent examiners exclusively analyzed the volume data sets without any knowledge of the patients’ histories, clinical findings or results of other imaging modalities. Furthermore, they had no information about the proportion of BI-RADS®-US 1, 2 and 5 cases in the database.The following standard procedure was applied to the systematic analysis of the ABVS data: Initially, the entire volume was explored in the coronal plane moving slowly (i.e. in thin slices) from the skin to the chest wall. During this process, the examiner marked all mass lesions with the system’s default tool. Next, the examiner evaluated all of the selected lesions by displaying them in the sagittal and axial planes. Finally, the examiner assigned the lesions a category according to the ACR BI-RADS®-US system. Overall interpretation times usually range from 4 to 10 min per case.Despite the fact that the examiner knew that there were no BI-RADS®-US 0, 3 or 4 cases in the database, he was allowed to categorize lesions as BI-RADS®-US 0, 3 or 4 whenever he requested a second-look ultrasound in order to further assess uncertain lesions.When a second-look ultrasound was requested for a lesion that eventually turned out to be benign, the result of the AVBS examination was defined as “non-concordant” . On the other hand, when a second-look ultrasound was requested for a lesion that turned out to be malignant, the result was classified as “concordant” (true-positive) because the cancer could then be correctly detected in the subsequent conventional ultrasound Table .Microsoft® Office Excel® 2007 (Microsoft Corporation) was used for data collection.Statistical analysis was performed by the author SW using MedCalc® 7.6 statistical software  and validated by the other authors.In order to assess the diagnostic performance of the ABVS, we calculated sensitivity, specificity and accuracy for both examiners and used the Z-Test to compare the performance of examiner 1 with examiner 2. As our study population did not reflect the real prevalence of breast cancer, the positive and negative predictive values were estimated based on the Bayesian theorem using the reported prevalence of malignancies in screening collectives . For theThe statistical analysis of the extent of agreement between the two raters was based on Cohen’s Kappa test. For the interpretation of κ-values we used the magnitude guidelines published by Landis and Koch, who characterized the values of κ<0 as indicating no agreement, κ 0–0.20 slight, κ 0.21-0.40 fair, κ 0.41-0.60 moderate, κ 0.61-0.80 substantial, and κ 0.81-1 as almost perfect agreement [Furthermore, we assessed the correlation between the expected and the observed rate of second-look ultrasounds using the Chi-square test.Statistical significance was assumed as p < 0.05 for all tests.In our study population, the age ranged from 19 to 86 years (median 52 years). According to the BI-RADS®-US categorization, 52% (n = 52) of our cases were assigned as BI-RADS®-US 1, 30% (n = 30) had BI-RADS®-US 2 lesions and 18% (n = 18) of the cases had BI-RADS®-US 5 lesions. All BI-RADS®-US 5 lesions were confirmed with histological specimens. The mean tumor size for malignant and benign lesions was 22.0 mm (range 13 to 55) and 16.7 mm (range 8 to 36), respectively.The concordance between examiner 1 and examiner 2 concerning the correct clinical decision of whether the patient should undergo a control ultrasound due to a suspicious finding or whether the patient should be defined as healthy as there is no suspicious lesion is shown in , the inter-rater validity coefficient calculated to κ=0.31 (95% CI: 0.21-0.41). Focusing on the benign cases (n = 82), the conditional inter-rater validity coefficient was κ=0.18 (95% CI: 0.00-0.26), indicating slight agreement. Concerning the malignant cases, we found a Cohen’s Kappa value of κ=0.80 (95% CI: 0.61-1.00), indicating substantial to almost perfect agreement  and 100% (95% CI: 78.1-100.0), respectively. The diagnostic accuracy of the method was 71.0% (95% CI: 60.9-79.4) and 60.0% (95% CI: 49.7-69.5), respectively. The differences between examiner 1 and examiner 2 were statistically not significant. Nevertheless, specificity revealed to be quite low at 68.3% (95% CI: 57.0-77.9) and 51.2% (40.0-62.3), respectively, as there was a relevant number of requests for second-look ultrasounds or further examinations in the group of healthy patients after the evaluation of the ABVS data. As previously described in the methods, these cases had to be classified as false-positives if there was no cancer , the evaluation was considered “negative”. In this scenario, the accuracy increased to 79.0% 95% CI: 69.5-86.3), the specificity increased to 78.1% (95% CI: 67.3-86.1) and the sensitivity remained acceptably high at 83.3% (95% CI: 57.7-95.6) . However, the observed rate of second-look ultrasounds was significantly high, totaling 41 for examiner 1 and 58 for examiner 2, respectively (p < 0.001).Therefore, the rate of second-look ultrasounds in healthy women (i.e. BI-RADS®-US 1 and 2) was 31.7% for examiner 1 and 48.8% for examiner 2.Regarding the BI-RADS®-US 1 and 2 cases separately, there was a request for a second-look ultrasound in 33.3% and 53.3% (examiner 1 and examiner 2) of the women with BI-RADS®-US 2 lesions and in 30.8% and 46.2% of the women with no breast lesions at all (i.e. BI-RADS®-US 1).Based on our findings, we performed a model calculation to estimate how additional ABVS examinations could affect the detection rate of cancer in screening collectives.As described in the literature, the prevalence of occult cancer that can be detected by additional ultrasound in women who already underwent mammography and clinical examination can be estimated to be between 0.3% and 0.4% -12. TherUsing these numbers, the positive predictive value in a screening collective would calculate to 1.49% and the negative predictive value to 99.91%, respectively. Therefore, the new false-negative rate in a diagnostic setting using the ABVS would be about 0.09% instead of 0.4% Table .If 10,000 women were additionally screened with the ABVS, we could expect 2,219 positive scans resulting in the same number of second-look-ultrasounds, finally leading to 33 detected cancers. Therefore, one cancer would be detected in 67 second-look ultrasounds. 7,781 scans would be negative, and only seven cancers would be missed in this group Figure .Innovative features for existing medical imaging modalities and even entirely new imaging technologies are constantly being developed ,23. BoosThe focus of our current study was on the inter-rater agreement concerning the evaluation of ABVS data. We were able to demonstrate fair agreement for both the classification into the categories “positive/negative” (κ=0.36) and the detailed classification according to the BI-RADS® system (κ=0.27). Nevertheless, this performance could be improved. The conditional inter-rater validity coefficients for benign and malignant cases revealed that the concordance is almost perfect for the malignant cases (κ=0.80), but only slight for the benign cases (κ=0.18). These results correlate with the experience, that both examiners reached high sensitivity , but lower specificity (68.3% and 51.2%). In other words, the detection of malignant lesions can be precisely performed with the ABVS. Cancers are rarely missed with this technology. The examiners agree in those cases in which a suspicious lesion is present in the volume data. On the other hand, the examiners tend to suspect doubtful lesions even in the normal breast tissue of healthy women. Consequently, the ABVS has a low specificity and a high false-positive detection rate. This condition impairs the conditional inter-rater validity for the benign cases and leads to a high number of unnecessary second-look ultrasounds. In fact, when the ABVS system is used, we lose the ability to immediately further explore a questionable lesion by modifying factors such as compression, the orientation of the probe and the machine’s setting whilst acquiring the image in real-time. Ultrasound techniques such Doppler imaging or sonoelastography cannot be used either. Because the examiners cannot rely on standardized interpretation criteria or rules how to handle technical artifacts in the volume data, they seem to prefer false positive evaluation rather than missing a malignant lesion. Nevertheless, with accumulating data, growing experience and better diagnostic criteria this problem may be solved.The consequence of a false positive result is the performance of an unnecessary second-look ultrasound, which is expensive, time-consuming and frustrating for both the patient and the medical professional. However, this second-look ultrasound implies no direct harm for the patient. A false-negative result may have a more serious implication as the diagnosis of malignancy is delayed, with a potentially worse clinical outcome for the patient.In conclusion, as the false-negative rate is low we assume high patient safety and encourage the clinical use of the ABVS for breast diagnostics.There is only limited data in the literature describing the inter-observer concordance in lesion detection with the ABVS:In 2011, Shin et al. reported on 55 women with 145 breast masses who were examined with handheld ultrasound and the ABVS . Five raRecently, Golatta et al. published data on 84 single breast examinations in 42 women . Six breIn our analysis, we found fair agreement between the two examiners, which correlates with the latter results (κ = 0.27). However, more data is needed to evaluate the performance of the ABVS in breast imaging convincingly.Moreover, there are several reports comparing the ABVS with hand-held ultrasound ,28-31:The first detailed description of the technical background and performance of the ABVS was published by our study group in 2011 . In 2011In 2012, Lin et al. published data on 81 patients and compared ABVS to handheld ultrasound. The authors described a perfect sensitivity for both methods , high specificity  and, consequently, a high diagnostic accuracy  . This peIn the same year, Wang HY et al. studied 239 lesions in 213 women who were scheduled to undergo biopsy. In this study, ABVS was similar to hand-held ultrasound in terms of sensitivity , specificity , and accuracy  .Recently, Wang ZL et al. published data on 153 patients with 165 breast lesions. The patients underwent mammography, ABVS and hand-held ultrasound. The authors reported no significant differences between ABVS and conventional ultrasound concerning sensitivity , specificity , and accuracy  ,31.Compared to our data, there is a discrepancy mainly concerning the specificity, which was lower in our investigation Table . ActuallHence, the main difference of our study design in comparison to the above-mentioned studies is that we did not focus on the evaluation of known lesions alone, but also on the detection. Therefore, our examiners did not know whether there was a lesion in the particular volume or not. In the studies from the literature, the examiners were well aware, that there is definitely a lesion in the volume that requires biopsy (i.e. BI-RADS® 3 and above). This knowledge certainly has an influence on the evaluation of the lesion and the overall performance of the ABVS.Our interpretation: the almost perfect performance of the ABVS as described in the literature is only valid for collectives of women with already pre-diagnosed breast lesions. In realistic collectives that also involve women without breast lesions (i.e. BI-RADS®-US 1) or with clearly benign breast lesions (i.e. BI-RADS®-US 2), our data may be more convincing.Concerning the inter-observer agreement, Zhang et al. published data on 234 breast lesions from 208 patients who were examined with the ABVS . Zhang eFurthermore, there are reports in the literature about the ABVS that concentrate on the optimal scanning technique , the accAll previously mentioned studies have the essential limitation of unicentric design and small patient collectives. This limitation also applies to our current study. Future study concepts should include multicenter design and larger, well-defined patient populations as described elsewhere . FurtherMoreover, the technology of the ABVS has some limitations per se. As previously described in the literature, automated breast ultrasound is limited in women with macromastia and pronounced ptosis ,16. In aFurthermore, we presume that even with optimal scanning technique the peripheral areas of the breast parenchyma are not fully covered by the ABVS. Additionally, shadowing artifacts occur in the retroareolar region and to a certain extent in the remaining breast volume. Therefore, a certain proportion of breast parenchyma may be lost in the volume data. This may reduce the diagnostic potential in comparison to hand-held ultrasound.Finally, we have to discuss the question of which role the ABVS could play in future breast cancer diagnosis and breast cancer screening. Conventional hand-held breast ultrasound has a commonly accepted role as a complementary method to mammography, by adding to the diagnostic accuracy. Despite the well-known advantages, conventional breast ultrasound is a time-consuming, examiner-dependent and therefore costly procedure. Furthermore, breast cancer screening with additional breast ultrasound in low-risk women has not been established, as data concerning an effect on the overall survival is missing. Nevertheless, it is evident that breast ultrasound is capable of detecting previously occult cancers in women who had already undergone mammography and clinical examination ,9,11,12.We can imagine the ABVS being applied in breast cancer screening programs in the future and that additional examinations are offered to women at risk. However, we still have no data focusing on certain subgroups of patients .A prospective randomized controlled trial could answer the open questions. This trial should include patients with normal mammograms, some of whom will receive additional ABVS. The primary outcome variables would be the detection rate of breast cancer, the tumor stage and the overall survival of the two groups. Presumably, results could only be obtained if the collectives were sufficiently large, which makes it likewise unrealistic that a corresponding trial will actually be conducted.In conclusion, the ABVS has demonstrated high sensitivity and a fair inter-observer concordance in the evaluation of breast lesions. However, we have to overcome the problem of the high number of false-positive results in healthy women. Therefore, further prospective validation studies in larger collectives are necessary to define standard procedures in image acquisition and volume interpretation. Nevertheless, the ABVS is a reliable, technically mature and potentially beneficial imaging method. Therefore, we propose that the ABVS can be used in routine diagnostic procedures as an adjunct to mammography and conventional ultrasound, as previously described.The authors declare that they have no competing interest.SW and SG contributed to the conception and design of the study and SW performed the ultrasound examinations and data collection. SG and AF performed the independent evaluation of the ABVS data sets. SW contributed to the statistical analysis and the interpretation of the data. SW and PS contributed to the writing of the manuscript. FD and PH conducted final reviews of the manuscript and FD provided methodological advice. All authors read and approve the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2342/13/36/prepub


Background. The heart consists of various kinds of cell components. However, it has not been feasible to separately analyze the gene expression of individual components. The laser microdissection (LMD) method, a new technology to collect target cells from the microscopic regions, has been used for malignancies. We sought to establish a method to selectively collect the muscular and vascular regions from the heart sections and to compare the marker gene expressions with this method. Methods and Results. Frozen left ventricle sections were obtained from Wistar-Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHR-SP) at 24 weeks of age. Using the LMD method, the muscular and vascular regions were selectively collected under microscopic guidance. Real-time RT-PCR analysis showed that brain-type natriuretic peptide (BNP), a marker of cardiac myocytes, was expressed in the muscular samples, but not in the vascular samples, whereas α-smooth muscle actin, a marker of smooth muscle cells, was detected only in the vascular samples. Moreover, SHR-SP had significantly greater BNP upregulation than WKY (P < 0.05) in the muscular samples. Conclusions. The LMD method enabled us to separately collect the muscular and vascular samples from myocardial sections and to selectively evaluate mRNA expressions of the individual tissue component. The heart consists of the cardiac muscle, vasculature, and to a lesser extent interstitial infiltrating cells. It is considered that the gene expressions are separately regulated in these tissue components under physiological and diseased conditions , 2. AlthRecently, the laser microdissection (LMD) method has been developed to isolate specific microscopic regions from tissue samples and separately collect the specimens of interest, which enables us to selectively evaluate the mRNA expression levels in targeted cell clusters in the tissues, especially in malignant tissues . The regThe study protocol was reviewed and approved by the Animal Care and Treatment Committee of Kurume University. Male Wistar-Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHR-SP) were purchased from SLC  and housed under standard conditions of humidity, room temperature, and a 12 : 12-hour dark-light cycles. They were provided with free access to tap water and chow. μm in thickness) were mounted on ice-cold PEN-slides .At 24 weeks, blood pressure was measured using a tail-cuff sphygmomanometer , as described previously –8. The n4 μm2 were collected in each animal.Fresh cryosections were fixed in RNase-free-ethyl acetate (acetic acid : ethanol = 1 : 19) and stained with 0.05% toluidine blue dissolved in RNase-free distilled water. The regions of the cardiac muscle and intramyocardial arteries were identified based on microscopic observation and were separately isolated from the section using LMD6000 system (Leica Microsystems). To collect vascular samples, we consistently placed the laser cut line on the outside border of the medial VSMC layer . The isoα-smooth muscle actin (α-SMA), and β-actin were obtained from GE Health Care. Good amplification plots for target genes were shown in the muscular and vascular samples  according to the manufacturer's instructions. Electropherogram exhibited clear peaks for 18S and 28S ribosomal RNAs in the purified RNA samples . RNA was samples . Expresst-test was performed for comparison between two groups.Data were expressed as mean ± SD. Unpaired n = 5) and 252.5 ± 16.7 mmHg in SHR-SP (n = 3) (P < 0.001). Percentage of Left ventricular fractional shortening was 26.8 ± 4.1% in WKY (n = 5) and 29.8 ± 5.2% in SHR-SP (n = 3) (no significance). We present one example of real-time RT-PCR analysis in WKY, which showed BNP mRNA expression in the muscular samples, but not in the vascular samples (α-SMA expression was found exclusively in the vascular samples. Next, we compared the expression levels of BNP, a molecular marker of cardiac myocyte hypertrophy, between WKY and SHR-SP (At 24 weeks of age, systolic blood pressure was 120.5 ± 12.2 mmHg in WKY ( samples . In contd SHR-SP . SHR-SP α-SMA, was expressed only in the vascular samples. Moreover, hypertrophic gene upregulation, as assessed by BNP expression, was significantly greater in the muscular samples obtained from SHR-SP than from WKY.The present study demonstrated that the LMD method enabled us to selectively collect myocardium and intramyocardial arteries from the heart section. Selective sampling was verified because the expression of cardiac myocyte-specific gene marker, BNP, was detected exclusively in the muscular samples, whereas the VSMC-specific marker, Recently, it has been reported that the LMD method is useful for selective sampling of the arterial component from the surrounding tissues, for example, the isolation of the arterial lesions in the lung specimen of patients with familiar pulmonary hypertension  and the μm. This finding was in line with the previous studies demonstrating that relatively small vascular samples, such as intrapulmonary arteries at a size between 50 and 200 μm and intrarenal arterioles with a diameter of approximately 100 μm, were selectively isolated using the LMD method [α-SMA were not detected in the vascular and muscular samples, respectively (As shown in D method , 17. In ectively . Moreoveectively . These fIn conclusion, the LMD method enabled us to separately collect the muscular and vascular samples from the myocardial sections and to selectively evaluate the mRNA expression changes in individual tissue component.


In this study, a combination of recombinant adenoviral p53 (rAd-p53) gene therapy and intra-arterial delivery of chemotherapeutic agents for treatment of oral squamous cell carcinoma was evaluated.In total, 99 patients with stage III or IV oral carcinoma who had refused or were ineligible for surgery were enrolled in a randomized, placebo-controlled, double-blind, phase III clinical trial. They were randomly assigned to group I , group II , or group III .P = 0.006). The rate of non-responders in group I was significantly lower than that in groups II and III (P < 0.020). A log-rank test for survival rate indicated that group I had a significantly higher survival rate than group III (P = 0.019). The survival rate of patients with stage III but not stage IV oral cancer was significantly higher in group I than in group III . The survival rate of patients with stage IV did not differ significantly among the three groups. Or the 99 patients, 63 patients experienced adverse events of either transient flu-like symptoms or bone marrow suppression, while 13 patients had both these conditions together. No replication-deficient virus was detected in patient serum, urine, or sputum. rAd-p53 treatment increased Bax expression in the primary tumor of 80% of patients, as shown by immunohistochemical staining.The median length of follow-up was 36 months . During follow-up, 16 patients in group I, 20 in group II, and 22 in group III died. Group I (48.5%) had a higher complete response rate than groups II (16.7%) and III (17.2%) . Oral cancer is a major global health problem with 300,000 new cases diagnosed each year. Alcohol and tobacco remain prominent etiologic factors in oral cancer , but higGene therapy with wild-type human tumor suppressor p53 (wt-p53) is a promising treatment strategy for some malignant tumors -12. The Several studies have examined the use of selective intra-arterial infusion of chemotherapy for head and neck cancers -39, and This randomized, placebo-controlled, double-blind, phase III pilot study of intra-arterial rAd-p53 with or without chemotherapy recruited 248 patients with advanced oral SCC between 2003 and 2007 from a single center . The study center was not a specialist referral center. Diagnosis of advanced disease (stages III and IV) was performed by three independent physicians experienced in oral and craniofacial surgery using World Health Organization (WHO) head and neck malignant tumor classification criteria, including patients’ clinical performance, clinical examination, and computed tomography/magnetic resonance imaging (CT/MRI). These stage III or IV oral cancer patients were ethnic Chinese from five provinces in southwest China, reflecting a wide geographic area, and they had refused or were not eligible for surgical treatment.Of the patients assessed, 149 patients were excluded for one or more of the following reasons: 1) systemic disease; 2) adenocarcinoma; 3) history of other treatments; 4) financial or logistical barriers to completing the trial; and 5) refusal of informed consent. Specifically, 68 patients were excluded for severe systemic disease ; 33 patients had received previous cancer-related therapy; 12 patients refused treatment for financial reasons; 17 patients had adenocarcinomas; and 19 patients did not give informed consent.This left 99 treatment-naive patients with advanced SCC  to be enrolled in the study, and they were randomly assigned to three treatment groups: group I ; group II ; and group III . Patients were randomized to the groups in a 1:1:1 ratio by using SAS 9.0 to generate a list of sequential numbers randomly from a permuted-block randomization procedure with a block size of 9 . Group III received the current standard chemotherapeutic drugs used as non-surgical treatment in our institution, with the modification of intra-arterial instead of intravenous infusion. A flow diagram of the study is shown in Figure No patients had undergone prior cancer treatment, and none had uncontrolled infections or immunodeficiency disease. Patients returned for periodic post-treatment physical examination, with follow-up by phone and mail, reflecting our standard practice.All enrolled patients provided written informed consent. The study was approved by the Institutional Review Board of State Key Laboratory of Oral Disease, West China School of Stomatology, Sichuan University, and was performed in accordance with Good Clinical Practice (GCP) guidelines for clinical trials, and according to the tenets of the Declaration of Helsinki.The recombinant adenoviral p53  is a recombinant human serotype 5 adenovirus in which the E1 region is replaced by a human wild-type p53 expression cassette ,43. The 12 virus particles (vp) per 2 ml ampoule. Frozen rAd-p53 was thawed, diluted in 8 ml normal 0.9% NaCl solution  at room temperature, and 10 ml were administered over a period of 20 minutes within 1 hour after thawing and dilution. Chemotherapeutic agents for treatment of SCC were carboplatin, bleomycin, and methotrexate. Intra-arterial infusion protocol  was identical in all three groups for active or placebo treatment. Saline solution was used for placebo infusions in groups II (placebo chemotherapy) and III (placebo rAd-p53).Recombinant adenoviral p53 was stored at -70°C at a concentration of 1 × 10The temporal region was anesthetized locally, and a long-term catheter was advanced retrogradely into the superficial temporal artery until the tip reached the opening of the main feeder artery arising from the external carotid artery. After catheterization was confirmed by infusion of methylene blue tracer, an indwelling arterial pump was implanted subcutaneously in the temporal region. Patients with advanced primary tumors that had spread to the contralateral side received bilateral catheterization. All treatments , were administered by means of an arterial pump. The same indwelling arterial pump was used to deliver gene therapy (20 minutes) and/or chemotherapy . The patients’ vital signs were monitored during administration.12 or 2 × 1012 vp per patient for unilateral or bilateral catheters, respectively. The rAd-p53 doses remained the same throughout the study. Patients were closely monitored for 4 hours after each rAd-p53 administration. Patients in group III received placebo rAd-p53  using this protocol, while those in group I received two courses of combination chemotherapy identical to that given to group III, and those in group ii received placebo chemotherapy  using the identical protocol. The first combination chemotherapy began 2 days after the first rAd-p53 infusion. The treatment periods were separated by a 7-day break. After infusion, the catheter was filled with 2000 U heparin to prevent coagulation.Patients in groups I and II received 10 cycles of rAd-p53 infusion within 6 weeks. The selected dose of rAd-p53 was delivered over 20 minutes via 10 ml syringes once every 4 days. The rAd-p53 doses were 1 × 10At the end of the study, some patients had become potential candidates for surgery or radiotherapy. Patients who experienced post-treatment recurrence during follow-up were given additional radiotherapy for salvage. To avoid a confounding effect of surgery on the outcomes of the three groups, post-treatment outcomes were assessed prior to local management or salvage therapy. Thus, the post-treatment outcomes of this study were not affected by these subsequent interventions.We evaluated the therapeutic responses of the primary tumor lesion by clinical examination (for lip carcinomas), or by CT or MRI . Once the initial scan revealed response, a follow-up scan was performed within 1 month to verify response. Treatment results were assessed as follows: 1) CR: disappearance of all tumor masses for at least 1 month; 2) partial response (PR): a decrease of 50% or more in the diameter product (largest diameter × perpendicular diameter) of the measurable tumor for at least 1 month; 3) stable disease (SD): a decrease of less than 50% or an increase of less than 25% in the diameter of the lesion; and 4) progressive disease (PD): an increase of greater than 25% in the diameter product, or the development of a new lesion. Toxicities and adverse clinical events, such as fever, chill, nausea, diarrhea, vomiting, fatigue, and myalgia, were evaluated and recorded, in accordance with the guidelines from the National Institute for the Control of Pharmaceutical and Biological Products .Patients in groups I and II had laboratory, hematology, and blood chemistry testing performed before treatment and within 24 hours after every rAd-p53 administration. Patients in groups I and III underwent the above examinations on the final day of each period of chemotherapy. Urine, stool, and serum samples were collected from patients in groups I and II. Serum samples were tested for the presence of anti-rAd-p53 immunoglobulin G. A cytopathic effect assay was used to detect the amount of vector disseminated in body fluids. Serum samples were analyzed for presence of anti-rAd-p53 antibody and vector within 24 hours and at 14 days after the initial infusion of rAd-p53, and at 1 month after the final rAd-p53 infusion.Biopsy specimens were taken within 3 days prior to receipt of rAd-p53 treatment and at 3 days after treatment. The specimens were immediately fixed in 10% formalin, blocked in paraffin wax, and cut into 5-μm serial sections. Sections were examined using hematoxylin and eosin staining. Adjacent sections were evaluated for expression of wt-p53, Bax (a protein induced by p53), and Bcl-2 (a protein that is down-regulated by p53) by immunohistochemistry using specific antibodies. Immunostaining of carcinoma cells was assessed qualitatively and semi-quantitatively. Semi-quantitative analysis scored the staining into the following four classes: if less than 10% of cells were labeled, it was scored as -; 0% and less than 25% staining as +, between 25% and less than 50% staining as ++, and 50% or greater staining as +++. Immunostaining of the samples was assessed by two independent blinded observers. Variations between the two observers were determined to be less than 5%. After serial dilution of serum, the anti-p53 antibody titers were detected by ELISA assay.2 test or Fisher’s exact test. Moreover, ordinal data  stage, Bcl-2 expression, and tumor grading) was determined using the Kruskal-Wallis test. The paired t-test was used to assess the difference in minimum inhibitory dilution  and counts of whole blood cells, lymphocytes, neutrophil and platelets before and after treatment. Kaplan-Meier curves with log-rank tests were created to assess the survival rates for the three groups. All statistical assessments were two-sided. and a P-value of 0.05 was set as significant. Statistical analyses were performed using SPSS 15.0 statistical software .Continuous variables in the three treatment groups were presented as mean ± standard deviation (SD) and compared using analysis of variance (ANOVA). Multiple comparisons between groups were performed by using the Bonferroni procedure with a type I error adjustment. Categorical variables were presented as numbers (percentages) and analyzed by the χIn this randomized, placebo-controlled, double-blind, phase III clinical trial, 99 patients with stage III or IV oral SCC were randomly assigned to group I , group II , or group III . Baseline characteristics of the three groups were similar (Table P < 0.020). The non-responder rate in group I patients with stage IV disease was 17.4% versus 50% in both groups II and III (P = 0.028). Few of the patients with stage III disease were non-responders, and the non-responder rates between the groups were not significantly different (P = 0.645).The primary lesions of 58 out of 92 patients responded to therapy, including 26 with CR and 32 with PD. The CR rate and the SD and PD rates were significantly different between the groups  . However, the survival rate of patients with stage IV did not significantly differ between the three groups .The median length of follow-up was 36 months (range 3 to 86 months). During follow-up, 16 patients in group I, 20 in group II, and 22 in group III died. There were three patients in group I, six in group II, and then in group III with post-treatment recurrence. Survival was evaluated for the entire population, and then for the patients with stage III or stage IV disease. A log-rank test for survival rate comparing the three groups indicated that group I had a significantly higher survival rate than group III . Antibody titers increased after administration of subsequent cycles of treatment; however, the anti-tumor activity of rAd-p53 was not affected by the neutralizing antibodies. No replication-deficient virus was detected in serum, urine, or sputum, suggesting that intra-arterial infusion did not result in systemic distribution.An example of a patient with CR from group I is presented in Figure P = 0.051). In addition, gene therapy alone (group II) did not induce bone marrow suppression, suggesting that this side effect was due to the chemotherapy.The most common treatment-related toxicities were transient flu-like symptoms (66 events) and bone marrow suppression 23 events). The majority of all groups experienced flu-like symptoms, especially the groups receiving rAd-p53 (Table 3 events.P < 0.05). Patients receiving chemotherapy showed significant declines in whole blood cell counts due to bone marrow suppression . A change in WBC count was not found in the group II patients  and Bcl-2 (a protein that is down-regulated by p53) . We examP = 0.787), and two patients received both surgery and radiotherapy. Thus, survival rates and other outcomes were related to the study treatments, and were not influenced by other interventions. Immunohistochemical analysis of biopsy specimens showed increased levels of staining for wt-p53 protein in those patients who received infusion of rAd-p53. Exogenous p53 protein produced by rAd-p53 uptake, and expression in the primary lesion appeared to be functionally active in the approximately 80% of patients who had higher Bax and lower Bcl-2 expression in the post-treatment primary tumor samples. Our results emphasize that rAd-p53 activity is intimately related to outcome. These findings are similar to those of Li et al., who observed an increase in p21 in 14 of 22 rAd-p53-treated tumor samples, a decline in bcl-2 staining in 8 of 22 samples, and detectable apoptosis  staining) in 18 of 22 samples [et al. [This single-center, randomized, placebo-controlled, double-blind, phase III clinical study showed that intra-arterial infusion of rAd-p53 and chemotherapy is associated with significantly better outcomes than either treatment alone, with a three-fold advantage in CR rate of the primary tumor mass. The survival rate was significantly higher in group I patients  with UICC III oral cancer. Patients selected for this study did not undergo prior surgery or radiotherapy, and treatment outcomes were recorded before any post-treatment local management by surgery. For complete disclosure, we state that after recording of all outcomes, the percentage of patients who underwent surgery was similar in group I , group II  and group III   to rAd-p53 therapy was associated with p53 status . High exet al. [Compared with previous studies using intratumoral or intravenous infusion of rAd-p53 ,11,36, tet al. ,11. Direet al. . Third, et al. , improveet al. [The combination of intra-arterial rAd-p53 and chemotherapy showed a significantly greater treatment response and survival benefit compared with either treatment alone, consistent with a synergistic interaction. Combination of rAd-p53 with other forms of treatment has also significantly improved CR and patient survival. Weinrib et al.  suggesteet al. . If wt-pet al. , which mResults from other trials of rAd-p53 therapy have shown that rAd-p53 treatment appears safe and well-tolerated ,15,36,48This study had several limitations. Although larger than previous investigations ,11, thisIn summary, the combination of gene therapy (rAD-p53) and chemotherapy produced significantly higher CR rates than either gene therapy alone or chemotherapy alone. These findings are consistent with a synergistic interaction, which has also been described in publications with other types of tumors. Interestingly, no significant adverse effects were found, and bone marrow suppression was significantly less common in group I (gene therapy plus chemotherapy) than in group III . This study also demonstrated that the p53 protein delivered by gene therapy is functionally active in 80% of cases . This pilot study provides the proof of benefits of intra-arterial infusion for cancer gene therapy. These results are noteworthy, and suggest that intra-arterial infusion of combined rAd-p53 and chemotherapy is a viable strategy for the management of oral SCC, and may represent an alternative to chemoradiotherapy and surgery, which is the current standard of care.®).None of the authors have any conflict of interest with the manufacturer of recombinant adenoviral p53 (GendicineYL was responsible for study concepts, definition of intellectual content, clinical, and experimental studies, and manuscript preparation. L-JL is the guarantor of the integrity of the entire study, and was responsible for study design, manuscript editing, and review. L-JW was responsible for clinical studies, data analysis, and statistical analysis. ZZ was responsible for clinical studies, manuscript editing, and review. NG was responsible for clinical studies, manuscript editing, and review. C-YL was responsible for literature research, experimental studies, and data analysis. Y-DH was responsible for experimental studies, data analysis, and manuscript preparation. BH was responsible for clinical studies and statistical analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/12/16/prepub


Puumala hantavirus (PUUV) causes a hemorrhagic fever with renal syndrome called nephropathia epidemica (NE). The aim of the present study was to evaluate plasma cell-free DNA (cf-DNA) levels and urinary cf-DNA excretion in acute NE as well as their associations with the severity of the disease.Total plasma cf-DNA was quantified directly in plasma of 61 patients and urine of 20 patients with acute NE. We also carried out a qualitative high-sensitivity lab-on-a-chip DNA assay in 20 patients to elucidate the appearance of cf-DNA in plasma and urine.vs. median 0.77 µg/ml, range 0.55–0.99 µg/ml, P<0.001). The maximum plasma cf-DNA levels correlated positively with maximum blood leukocyte count  and the length of hospital stay , and inversely with minimum blood platelet count . Qualitative analysis of plasma cf-DNA revealed that in most of the patients cf-DNA displayed a low-molecular weight appearance, corresponding to the size of apoptotic DNA (150–200 bp). The visually graded maximum cf-DNA band intensity correlated positively with the maximum quantity of total plasma cf-DNA . Maximum urinary excretion of cf-DNA in turn was not markedly increased during the acute phase of NE and did not correlate with any of the variables reflecting severity of the disease or with the maximum plasma cf-DNA level.The maximum plasma cf-DNA values taken during acute NE were significantly higher than the control values taken after the hospitalization period (median 1.33 µg/ml, range 0.94–3.29 µg/ml The plasma levels of cf-DNA are elevated during acute PUUV infection and correlate with the apoptotic cf-DNA-band intensity. The plasma cf-DNA concentration correlates with some variables reflecting the severity of the disease. The urinary excretion of cf-DNA does not reflect the degree of inflammation in the kidney. Myodes glareolushttp://www3.ktl.fi).Puumala hantavirus (PUUV) is a rodent-borne zoonotic virus carried by the bank vole, The severity of PUUV infection varies from subclinical disease to rare fatal cases The pathogenesis of NE is not completely understood. An important feature in hantaviral infections is capillary leakage due to increased capillary permeability. The mechanisms behind this phenomenon are unclear, but complement activation may be involved Circulating cell-free DNA (cf-DNA) has recently been studied in various acute and chronic clinical disorders. Elevated levels of cf-DNA have been reported in cancer, autoimmune diseases, stroke, myocardial infarction, trauma patients and sepsis Studies on plasma cf-DNA in viral infections are scarce and urine levels of cf-DNA have not previously been studied in infectious diseases. In the present study, our aim was to assess cf-DNA plasma levels and urine excretion in patients with acute PUUV infection. We aimed to evaluate the associations of plasma cf-DNA levels with the clinical severity of the disease. We also wanted to assess whether the excretion of cf-DNA is associated with the severity of the infection or renal insufficiency. In addition to measuring total cf-DNA levels directly in plasma and urine, we carried out a qualitative high-sensitivity lab-on-a-chip DNA assay to elucidate the appearance of cf-DNA in plasma and urine during the course of the infection.The study cohort consisted of 61 prospectively collected consecutive adult patients with acute NE. The diagnosis of acute PUUV infection was serologically confirmed in all cases The following diseases before NE were prevalent in 24 (39%) patients: essential hypertension in eight; dyslipidemia in six; atrial fibrillation in three; coronary artery disease, bronchial asthma, hypothyreosis, chronic inflammatory bowel disease, and hyperplasia of the prostate in two patients each; diabetes mellitus, sick sinus syndrome treated with pacemaker, Sjögren's syndrome, mitral valve disease, epilepsy, fibromyalgia, sarcoidosis, multiple sclerosis, operated atrial septal defect, and operated melanoma were present in one patient each.All subjects gave an informed consent before participation and the study was approved by the Ethics Committee of Tampere University Hospital.All 61 patients were examined during the acute phase of NE. A detailed past and current medical history was obtained, and a careful physical examination was performed. Blood samples were collected between 7:30–9:30 in the morning for up to five consecutive days after hospitalization. They were used for the analysis of plasma cf-DNA, PTX3, IL-6 (from 48 patients), CRP, and creatinine, as well as serum kynurenine (Kyn) and tryptophan (Trp) levels, and also for the blood cell counts. Other blood samples were taken according to the clinical needs of the patient.The nightly urine collection was performed during two consecutive nights after hospitalization. The nightly collection period was set from the time of the last voiding at bedtime until the last voiding on rising. After completion, volume was measured and timing was recorded for the collection period.The highest and the lowest values of the various variables measured during hospitalization for each patient were designated as the maximum and minimum values.One to three chest radiographs were obtained from 38 patients (62%).Fifty-three (87%) of the 61 patients were also studied at the out-patient clinic four weeks after the hospital period. The plasma and urine samples taken four weeks after the hospital treatment were regarded and assessed as control/recovery samples.The cf-DNA analyses were performed afterwards from frozen samples stored at −70°C. The amount of total cf-DNA was determined directly in plasma and urine without any DNA purification step, using the Quant-iT™ high-sensitivity DNA assay kit and a Qubit® fluorometer  following the manufacturer's instructions. Plasma samples were analysed in duplicate and the mean of the two values was used as the final value. The assessed intra-day variation coefficients at the mean plasma cf-DNA levels of 0.673 µg/ml, 0.876 µg/ml and 1.59 µg/ml were 4.2%, 1.0% and 4.1%, respectively. The corresponding inter-day variation coefficients were 5.5%, 4.3% and 6.6%. Total cf-DNA in urine was measured in 20/61 patients with quadruple measurements in which the mean of the four values was used as the final value. The assessed intra-day variation coefficients at the mean urine levels of 0.307 µg/ml, 0.769 µg/ml and 1.13 µg/ml were 5.0%, 4.8% and 3.5%, respectively. The corresponding inter-day variation coefficients were 10.0%, 6.5% and 10.2%. Timed overnight urinary excretion of cf-DNA was calculated as follows: /(time span).Qualitative analysis of plasma and urine cf-DNA was performed for randomly selected 10 patients with and without renal insufficiency . Plasma and urine cf-DNA was extracted using the NucleoSpin® Plasma XS Kit , designed for isolation of low-molecular-weight (50–1000 bp) cf-DNA. Cf-DNA isolation was performed according to the manufacturer's instructions following the high-sensitivity protocol. Extracted cf-DNA samples were analyzed with the High Sensitivity DNA assay kit and an Agilent 2100 Bioanalyzer equipped with Expert 2100 software according to the manufacturer's instructions . Agilent 2100 Bioanalyzer uses a lab-on-a-chip technology to perform gel electrophoresis; nucleic acids are separated analogously to a capillary electrophoresis and normalized to a ladder and two DNA markers, after which the software automatically calculates the size of each band. For each plasma sample, the appearance and intensity of low-molecular weight cf-DNA was estimated visually and graded as follows: 1 = no visible cf-DNA or extremely weak band intensity, 2 = intermediate band intensity, 3 = strong band intensity. The researcher responsible for analysing and grading the cf-DNA samples was blinded for the clinical data of the patients. The appearance of cf-DNA in urine was analyzed descriptively.Plasma CRP and creatinine levels were analyzed using Cobas Integra analyzer . Blood cell count was completed by hematological cell counters by Bayer. Plasma IL-6 concentrations were determined as previously described U- test. Categorical data were analyzed by the x2 test or the Fisher's exact test, as appropriate. Correlations were calculated by the Spearman's rank correlation test. Wilcoxon's test was used to compare two related samples. All tests were two-sided, and statistically significant P-values are given. All analyses were made with the SPSS (version 18) statistical software package.In order to describe the data, medians and ranges were given for continuous variables and numbers and percentages for categorical variables. Groups were compared using the Mann-Whitney The clinical characteristics of the 61 patients are shown in vs. median 0.77 µg/ml, range 0.55–0.99 µg/ml, P<0.001). Maximum urinary excretion of cf-DNA was not increased during the acute phase of NE when compared with the control values after the hospitalization . The control values were taken median 41 (range 18–83) days after the onset of fever.The maximum total plasma cf-DNA values taken during acute NE were significantly higher than the control values taken after the hospitalization period , maximum plasma PTX3 levels , and the length of hospital stay . There was also an inverse correlation between maximum plasma cf-DNA levels and minimum blood platelet count . The maximum plasma cf-DNA levels did not correlate with maximum plasma creatinine levels or minimum urinary output . Neither did the maximum cf-DNA levels correlate with minimum hematocrit, maximum plasma CRP, IL-6 or serum IDO levels . There was no correlation between plasma cf-DNA and age .The maximum plasma cf-DNA levels correlated positively with maximum blood leukocyte count .The maximum urinary excretion of cf-DNA did not correlate with any of the variables reflecting severity of the disease, and it did not correlate with age or maximum plasma cf-DNA, either (data not shown).r = 0.513, P = 0.021). However, the maximum cf-DNA band intensity did not correlate with any of the clinical or laboratory parameters. In control samples, which were taken four weeks after the hospital period, the low-molecular weight cf-DNA band was either completely absent or markedly weakened in all patients  . This phpatients .Qualitative analysis of urine-cf-DNA revealed that in contrast to plasma, no distinguishable low-molecular weight (150–200 bp) pattern of cf-DNA was detected during the acute phase of the disease, with the exception of two patients with renal insufficiency (patients no 5 and 7 with maximum plasma creatinine >370 µmol/l) . In addiThe data presented here show that plasma cf-DNA levels are elevated during acute PUUV infection. The cf-DNA values measured during the acute phase were markedly higher than the values measured after the hospitalization. Previously, in bacteremia patients the median maximum cf-DNA levels were 2.03 µg/ml in nonsurvivors and 1.26 µg/ml in survivors Qualitative analysis of plasma cf-DNA revealed that, during the acute phase of infection, cf-DNA displayed a predominance of low-molecular weight and apoptotic (150–200 bp) appearance, whereas after recovery, such cf-DNA pattern was not observed in any of the patients. It is thus likely that the detected low-molecular weight cf-DNA originated from apoptotic cells in the course of the acute phase of the disease. In fact, results from a recent study in PUUV infected patients suggest that acute hantavirus infection is associated with immune reaction-induced renal tissue damage K-ras sequences in the transrenal urine cf-DNA The observation that the correlates for maximal total urinary cf-DNA excretion were different from those of the maximum plasma cf-DNA concentration suggest that the amount of cf-DNA in urine may not be clinically relevant and the excretion of cf-DNA does not reflect the degree of inflammation in the kidneys in acute NE. Our findings are corroborated by previous results which have demonstrated that in hematopoietic stem cell transplant patients, the quantity of donor-derived cf-DNA in urine does not correlate with that of plasma and that the predominant cf-DNA fragment size differs between plasma and urine Previously cf-DNA has been investigated in a variety of clinical conditions. However, studies concerning infections other than sepsis are scarce. In septic patients, there are studies showing elevated cf-DNA levels that also predict outcome In conclusion, the plasma levels of cf-DNA are elevated during acute PUUV infection and correlate with the apoptotic band intensity. The total plasma cf-DNA concentration correlates with some variables reflecting the severity of the disease. The urinary excretion of cf-DNA does not reflect the degree of inflammation in the kidney.


A broad spectrum of pathogens is causative for respiratory tract infections, but symptoms are mostly similar. Therefore, the identification of the causative viruses and bacteria is only feasible using multiplex PCR or several monoplex PCR tests in parallel.The analytical sensitivity of three multiplex PCR assays, RespiFinder-19, RespiFinder-SMART-22 and xTAG-Respiratory-Virus-Panel-Fast-Assay (RVP), were compared to monoplex real-time PCR with quantified standardized control material. All assays include the most common respiratory pathogens.1 to 105 copies/ml. Concordant results were received for rhinovirus, whereas the RVP detected influenzavirus, RSV and hMPV more frequently in low concentrations. The RespiFinder-19 and the RespiFinder-SMART-22 showed a higher analytical sensitivity for adenoviruses and coronaviruses, whereas the RVP was incapable to detect adenovirus and coronavirus in concentrations of 104 copies/ml. The RespiFinder-19 and RespiFinder-SMART-22A did not detect influenzaviruses (104 copies/ml) and RSV (103 copies/ml). The detection of all 13 viruses in one sample was only achieved using monoplex PCR. To analyze possible competitive amplification reactions between the different viruses, samples were further inoculated with only 4 different viruses in one sample. Compared to the detection of 13 viruses in parallel, only a few differences were found.To compare the analytical sensitivity of the multiplex assays, samples were inoculated with 13 different quantified viruses in the range of 10The incidence of respiratory viruses was compared in tracheal secretion (TS) samples (n = 100) of mechanically ventilated patients in winter (n = 50) and summer (n = 50). In winter, respiratory viruses were detected in 32 TS samples (64%) by RespiFinder-19, whereas the detection rate with RVP was only 22%. The most frequent viruses were adenovirus (32%) and PIV-2 (20%). Multiple infections were detected in 16 TS samples (32%) by RespiFinder-19. Fewer infections were found in summer . All positive results were verified using monoplex PCR.Multiplex PCR tests have a broad spectrum of pathogens to test at a time. Analysis of multiple inoculated samples revealed a different focus of the detected virus types by the three assays. Analysis of clinical samples showed a high concordance of detected viruses by the RespiFinder-19 compared to monoplex tests. Acute respiratory tract infections are the most widespread type of infection in adults and children and are responsible for a considerable morbidity and mortality worldwide. A high rate of respiratory tract infections is caused by viruses (approximately 80%) ,2. WithiThis study presents the first comparison of the analytical sensitivity of three novel multiplex PCR methods, the RespiFinder-19 assay, RespiFinder-SMART-22 assay  and the xTAG Respiratory Virus Panel Fast Assay , with quantified virus control material. The RespiFinder-19 assay, which is based on the multiplex ligation-dependent probe amplification analyzed by capillary electrophoresis, detects 15 respiratory viruses and 4 bacteria in one reaction . A furthThe aims of the present study were as follows: (a) the quantification of commercially available qualitative control material for (b) the evaluation of the performance and sensitivity of the three multiplex assays RespiFinder-19, RespiFinder-SMART-22 and RVP fast assay and (c) the applicability and performance of these multiplex PCR assays in a routine setting. Furthermore, this is the first screening study determining the incidence of infections with respiratory pathogens in a mechanically ventilated patient cohort developing an atypical pneumonia during post-operative monitoring using multiplex PCR assays.Legionella pneumophila, Pneumocytisjirovecii, Mycoplasma pneumoniae and Chlamydophilapneumoniae. The residual material was used for the comparative analysis with the RespiFinder-19 and RVP assay. The results of the performance evaluation were received retrospectively and had no influence on patient's therapy. All patients provided informed consent. For our study we did not need an ethical approval, because in paragraph §24 of the German Act on Medical Devices (in German: GesetzueberMedizinprodukte - MPG) an ethical approval is not required for clinical specimens without a separate invasive sampling.Tracheal secretion (TS) samples (n = 100) were collected in February  and July  2010 from mechanically ventilated non-immunocompromised patients . Patients were suspected of atypical pneumonia during postoperative monitoring after coronary artery bypass, heart or lung surgery. TS samples were initially analyzed for bacterial and viral pathogens with our routine diagnostic profile including CMV, The qualitative Respiratory Validation Panel Global  was quantified by real-time qPCR. External plasmid standards were used to determine the concentration of the different viruses. Plasmid standards of adenovirus , influenza A virus , RSV-A , enterovirus  and rhinovirus  were established [The virus controls of the NATRVP-2 are purified intact virus particles that have been chemically modified to render them non-infectious and refrigerator stable. Quantifications of viruses included in this panel were as follows: influenza A virus , RSV-A (5.12E + 05 copies/ml), adenovirus (4.63E + 05 copies/ml), rhinovirus (3.68E + 05 copies/ml), hMPV (2.31E + 05 copies/ml), PIV-1 (2.09E + 05 copies/ml), PIV-2 (1.19E + 05 copies/ml) and PIV-3 (4.99E + 05 copies/ml) . Accordingly, for the undiluted samples inoculated with 4 viruses, 22 μl of each virus were mixed and filled up with PBS to achieve also a final volume of 286 μl. Subsequently, 200 μl of the sample was extracted using the NucliSENSeasyMAG automated system with an elution volume of 55 μl.The total nucleic acid from inoculated samples and tracheal secretion samples (TS) was extracted using the NucliSENSeasyMAG automated system  according to the manufacturer’s instructions. Nucleic acids were extracted from 500 μl of TS samples and 200 μl of inoculated samples and were eluted in a final volume of 55 μl (routine diagnostic) or 100 μl elution buffer, respectively . The manufacturer of the Respi-Finder-19 and SMART-22 assays (Pathofinder) recommend an elution volume of 100 μl, whereas the manufacturer of the RVP assay recommend an elution volume of 55 μl. To avoid false negative results due to a deviation from the manufacturer instructions, we extracted residual material and used an elution volume of 100 μl.The nucleic acid extracts were tested using the RVP assay  according to the manufacturer’s instructions. The RVP employs a multiplex PCR with labelled primers and a single-step hybridization of PCR products to the fluorescent bead array. The detection was performed using the xMAP 100 IS instrument  and the analysis was performed using TDAS RVP FAST software .The RVP simultaneously detects influenza A virus , influenza B virus, RSV-A and -B, adenovirus, hMPV, PIV-1, -2, -3 and −4, coronaviruses 229E, NL63, OC43 and HKU1, picornavirus (enterovirus and rhinovirus) and human bocavirus. The assay also includes an internal positive control added to each specimen at the extraction step (phage MS2) and a positive run control that is added to each plate (phage lambda DNA) .The RespiFinder-19 and the RespiFinder-SMART-22  were used according to the manufacturer’s instructions except for the nucleic acid extraction of patient samples, because we use 500 μl of TS samples instead of 200 μl as recommend. Elution volumes were used as described previously. Briefly, the assays comprised a preamplification step, which combines reverse transcriptase and multiplex target amplification PCR, followed by a probe hybridization step, a probe ligation step and a probe amplification step. The RespiFinder-19 analyzes the amplified PCR products by capillary electrophoresis using a DNA analyzer , whereas the RespiFinder-SMART-22 analyzes by melt curve analysis on the RotorGene Q .Bordetella pertussis, Chlamydophila pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae). The RespiFinder-SMART-22 additionally detects coronavirus HKU1, enterovirus and bocavirus. Both assays also tests an internal positive control added to each specimen at the extraction stage  [The RespiFinder-19 simultaneously detects 15 respiratory viruses  and four bacteria (s virus) .Legionella pneumophila[PCR primers and probes were adapted as previously described: adenovirus , coronaveumophila. The baceumophila. A 89 bp4), 400 nM of target primers, 100 nM of the target probe, 200 nM of IC primers, 100 nM of the IC probe and 5 U of Invitrogen SuperScript Platinum Taq-enzym mix . A 203-bp PCR product of the bacteriophage MS2 replicase gene was added to the reaction mixture as an exogenous IC. PCR was performed on the RotorGene Q system  with a reverse transcription at 50°C for 15 min, preliminary denaturation at 95°C for 10 min, followed by 45 cycles of denaturation of 95°C for 15 s, annealing and extension at 60°C for 45 s, with a single fluorescence acquisition step at the end of the annealing step.RNA amplification of coronavirus , influenza A virus, RSV-A and rhinovirus was carried out in 0.2 ml tubes containing 45 μl reaction mix and 5 μl RNA extract. The reaction mix consisted of 2 × Invitrogen rnx-reaction mix . PCR was performed on the Rotor-Gene 3000 system  with an UNG activity step at 37°C for 5 min, preliminary denaturation at 95°C for 10 min, followed by 45 cycles of denaturation of 95°C for 15 s, annealing and extension at 65°C for 45 s, with a single fluorescence acquisition step at the end of the annealing step.DNA amplification of CMV, adenovirus and enterovirus was carried out in 0.2 ml tubes containing 45 μl reaction mix and 5 μl DNA extract. The reaction mix consisted of 10 × Taq buffer (including 50 mM Mg), 400 nM of each deoxynucleoside triphosphate, 500 nM of target primers, 200 nM of the target probe, 300 nM of IC primers, 100 nM of the IC probe, 0.01 U of Uracil-DNA Glycosylase  and 5 U of Legionella pneumophila PCR was carried out in glass capillaries containing 15 μl reaction mix and 5 μl DNA extract. The reaction mix consisted of 5 × FastStart DNA Master Plus Hybridization Probes , 200 nM of each target primer, 150 nM of the target probe, 300 nM of each IC primer for lambda, 100 nM of the IC probe and 0.01 U of UNG . PCR was performed on the LightCycler 2.0 system  with an UNG activity step at 37°C for 10 min, preliminary denaturation at 95°C for 10 min, followed by 45 cycles of denaturation of 95°C for 2 s, annealing at 55°C for 10 s and extension at 72°C for 15 s, with a single fluorescence acquisition step at the end of the annealing step, and a following melt analysis (95°C for 60 s and 40°C for 30 s).The The real-time PCR for hMPV, PIV-1, -2 and −3 was performed using LightMix assays  on the LightCycler 2.0 system according to the manufacturer’s instructions.CT) values, determined from six independent PCR runs. Key parameters for real-time PCR assays are shown in Table The cut-off value for the decision positive/negative was adjusted to <40 cycles. The specificity of all monoplex-real-time PCR assays was determined by the exclusion of cross-amplification with different bacterial or viral DNAs/RNAs . The analytical sensitivity was determined to be <10 copies/ml. The reproducibility of the assay was demonstrated by analyzing the inter-assay variation for the crossing threshold . The RVP showed the detection of influenzavirus A , respiratory syntical virus A and B , coronavirus OC43 (CoV OC43) and human metapneumovirus  even in a high dilution ratio (Table 4 copies/ml. Only the RespiFinder-SMART-22 detected PIV-2 at a concentration of 92 copies/ml). None of the three multiplex assays was capable of detecting all 13 viruses in parallel. This was only achieved using monoplex real-time PCR assay. CT values were shown in Table Virus control material, NATRVP2 (Zeptometrix), was quantified by external plasmid standards for a comparison of the analytical sensitivity of the RVP, RespiFinder-19 and RespiFinder-SMART-22 assays. Subsequently, PBS-buffer was inoculated with 13 different quantified viruses, or 4 viruses in different combinations, in the range of 104 copies/ml (panel 1) to 102 copies/ml . The RVP assay now detect AdV  and PIV-2 . The RVP was still incapable of detecting CoV 229E. However, more differences were found in the detection of the pathogens using different elution volumes for both RespiFinder assays. The company PathoFinder suggests an elution volume of 100 μl for the RespiFinder-19 and the RespiFinder-SMART-22 assay, whereas Abbott Molecular advises for the RVP assay an elution volume of 55 μl. The two assays of the company PathoFinder did not detect INF-A in samples which were extracted in 55 μl elution buffer, but detection was possible by using the advised extraction volume of 100 μl. The RespiFinder-19 detect INF-A at a concentration of 104 copies/ml (panel 1), whereas both assays detect INF-A at concentrations ranging from 102 -104 copies/ml (panel 2). The same observation was made for the detection of AdV at a concentration of 102 copies/ml (panel 1) with the RespiFinderSMART-22 by using the advised extraction volume of 100 μl. In contrast, the detection frequency of RSV-A virus was reduced for both RespiFinder assays analysing samples with 100 μl elution volume compared to 55 μl elution volume . No differences in the detection frequency were found for the RVP assay  and summer (July). The RespiFinder-SMART-22 as a further development of the RespiFinder-19 was not participating in this study, because it started before the RespiFinder-SMART-22 was commercially available. All positive results were verified with real-time PCR as monoplex analysis. TS samples were obtained routinely from mechanically ventilated patients after coronary artery bypass, heart or lung surgery who were suspected of atypical pneumonia during post-operative monitoring.L. pneumophila was detected by the RespiFinder-19 (2%). In order to confirm positive results obtained by RVP and RespiFinder, samples were additionally tested by monoplex real-time PCR with 100% concordance   from February, multiple infections were detected Table . One triThe detection of respiratory viruses by multiplex PCR has been described as an important tool for the identification of the pathogens in respiratory tract infections.But until today no comparison of these three multiplex PCR methods with quantified standardized virus control material was performed. Therefore, we quantified virus control material to compare the analytical sensitivity of three commercially available multiplex PCR methods, followed by evaluation of application for routine diagnosis with regard to hands-on-time, time-to-result, costs and accomplishment Table .1-102 copies/ml. CoV OC43 and HRV were detected equally also at low concentrations with the three assays, whereas PIV-1 and PIV-3 were not detected at all. The parallel analysis with monoplex real-time PCR assays showed as expected the highest analytical sensitivity: all viruses were detected in all concentrations and dilution factors.We observed that the RVP, the RespiFinder-19 and the RespiFinder-SMART-22 assay had a different focus of the detected virus types in the inoculated samples. The RVP showed an advanced detection of INF-A, RSV, and hMPV, whereas the RespiFinder-19 and the RespiFinder-SMART-22 showed an improved detection of CoV 229E and AdV. The RespiFinder-SMART-22 further showed an improved detection of PIV-2. Viruses were detected also at low concentrations of 10Our results indicated a possible competition for nucleotides, primer or enzymes between the different viruses in the detection of a high number of multiple infections (13 viruses in one assay). 13 viruses in one sample was the maximum demand on the multiplex PCR tests. This highly artificial experiment emphasizes the general methodological limitation of multiplex PCR assay, because nearly all multiplex assays performed terribly analyzing samples with a high number of parallel pathogens. Therefore, these results have to be interpreted with attention. In the routine clinical setting, a parallel infection with this high number of different viruses is uncommon up to impossible, and the clinical relevance of these results is initially arguable. The highest virus load in our patient cohort was a triple infection. This was also observed by other research groups . Due to et al.[et al.[et al.[et al. also described problems in the detection of co-infections with the RVP. Samples which include adenovirus and coronavirus were false-negative with the RVP. We observed these problems in samples, which were positive for CMV and adenovirus. The importance of viral co-infections remains uncertain [The analysis of 100 TS samples was done in parallel with the RVP and the RespiFinder-19. At that time, the RespiFinder-SMART-22 was not commercially available and later on, no residual extracted material was available for additional testing. For a comparison of the three multiplex assays with clinical samples further studies have to be done. This study indicated a higher clinical sensitivity of the RespiFinder-19 in the detection of virus infections in clinical samples with low concentrations in contrast to the RVP. Respiratory tract infections were found in 64% of the TS samples by RespiFinder-19. The RVP detected only 22% in the same samples, respectively. Gadsby et al. also desl.[et al. investigncertain -13. In sncertain . In thisThe interpretation of the generated data of the three assays has different levels of difficulty. Abbott Molecular supplies with the RVP a software tool (TDAS RVP FAST software), which analyze the generated data and provided automatically a positive or negative signal for the detection of the particular virus. The interpretation of the results of the RespiFinder-19 is more difficult, because the manufacture did not provide helping data for the analysis (e.g. threshold). So it is possible that low positive samples were missed. The further developed RespiFinder-SMART-22, has an improved analysis of the generated data with given melting points.http://www.medical-dpc.com/respvir) will allow a constant comparison of seasonal accumulations and chronological trends.In general, the comparison of the spectrum of pathogens detected in our patient cohort of mechanically ventilated patients with other studies (e.g. ) is hardFor the application of the three multiplex PCR methods in a routine diagnostic setting, the hands-on-time, the time-to-result, the costs and the accomplishment are also very important parameters. Table Study limitations: Unless samples were not analyzed in an adequate number of replicates to perform statistical analysis, our approach allow the comparison of the performance and the range of sensitivity of the three multiplex assays by analysis of inoculated samples with quantified virus material. Reported differences may be due to chance regarding a single consideration of individual results, however a tendency towards difference in lower or higher detection efficiencies by the different assays is observable.In conclusion, our study shows that before a multiplex PCR method is applied in routine diagnostics it has to balance out the analytical sensitivity between time-to-result, hands-on-time and the clinical relevance of the detected pathogens. The RespiFinder-19 has a higher analytical sensitivity than the RVP, but needs more than twice as long for a result compared to the RVP. The RespiFinder-SMART-22, as a further development provides a faster result, but has to been tested with routine samples in further studies.The authors declare that they have no competing interests.M.D.-R. conducted the experiments, analyzed the data and wrote the paper, T.V. coordinated the study, O.A. revising the manuscript critically for important intellectual content, C.K. and J.D. initiated the study, planned the experimental design and contributed to write the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/163/prepub